Cyclooxygenase-2 (COX-2) is a key enzyme of arachidonic acid metabolism and an important contributor to a range of acute and chronic human diseases. COX-2 inhibitors are antiinflammatory, analgesic, and cancer chemopreventive but lack the serious gastrointestinal side effects that plague traditional non-steroidal antiinflammatory drugs (NSAIDs). Our laboratory is interested in the structural basis of catalysis and inhibition of COX-2. We recently reported a fluorescence quenching technique for monitoring enzyme-inhibitor association and dissociation in real time and used it to demonstrate that the COX-2-selectivity of diarylheterocycle inhibitors is due to their very slow dissociation from the enzyme compared to COX-1. We propose to use fluorescence quenching to explore the role of residues in a side pocket of the cyclooxygenase active site and in the constriction that leads to the cyclooxygenase active site in the binding and release of diarylheterocycles from the enzyme. Studies of enzyme-substrate interactions led us to design a novel series of covalent and non-covalent COX-2 inhibitors. Conversion of carboxylic acid-containing NSAIDs into neutral ester or amide derivatives dramatically increases their selectivity for COX-2. We propose to use site-directed mutagenesis and X-ray crystallography to elucidate the molecular basis for interaction of COX-2 with a series of indomethacin amides and esters. Finally, our studies of COX-2 interactions with NSAID esters and amides led us to discover a novel substrate for COX-2 that may provide a new pathway of signal transduction. We propose to investigate the metabolism of 2- arachidonylglycerol by COX-2, the molecular basis for its enzyme specificity, and the ability of its endoperoxide metabolite to be converted into glyceryl eicosanoids. These investigations will provide important new insights into the function and inhibition of COX-2, a key enzyme of lipid signalling and human disease.